Layered collagen and ha scaffold suitable for osteochondral repair

ABSTRACT

The invention relates to a method for producing a multi-layer collagen scaffold. The method generally comprises the steps of: preparing a first suspension of collagen and freezing or lyophilizing the suspension to provide a first layer; optionally preparing a further suspension of collagen and adding the further suspension onto the layer formed in the previous step to form a further layer, and freezing or lyophilizing the layers, wherein when the layer formed in the previous step is formed by lyophilization the lyophilized layer is re-hydrated prior to addition of the next layer; optionally, repeating the aforementioned step to form one or more further layers; and preparing a final suspension of collagen and pouring the final suspension onto the uppermost layer to form a final layer, and freeze-drying the layers to form the multi-layer collagen composite scaffold.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 14/725,337 filed on May 29, 2015, which is acontinuation application of U.S. patent application Ser. No. 14/084,164filed on Nov. 19, 2013, now U.S. Pat. No. 9,072,815, issued on Jul. 7,2015, which is a divisional application of U.S. patent application Ser.No. 13/145,970 filed on Oct. 6, 2011, now U.S. Pat. No. 8,613,943,issued on Dec. 24, 2013, which is a national phase application under 35U.S.C. § 371 of International Application No. PCT/IE2010/000005, filedon Jan. 25, 2010, which claims priority to and the benefit of U.S.Provisional Application No. 61/147,006, filed Jan. 23, 2009 and EuropeanPatent Application No. 09151226.9, filed Jan. 23, 2009, the contents ofeach of which are incorporated herein by reference in their entireties.

INTRODUCTION

The invention relates to a method for producing a multi-layer collagenscaffold suitable for osteochondral defect repair. The invention alsorelates to a multi-layer collagen-composite scaffold, and uses thereofin osteochondral defect repair.

Articular cartilage is a highly specialised tissue found covering thesurfaces of the bony ends of all synovial joints in the human body. Itsfunction is to lubricate joint movement and absorb small shock impactswithin a joint. Articular cartilage is composed of 70-80% water, 9%aggrecan (the main water binding molecule within the collagen matrix),15% collagens (80% type II, 15% type IX and XI and 5% type III, VI, XII,XIV) and 3% cells (Aigner T, Stove J.; 2003).

The structure and composition of articular cartilage is highly orderedinto distinct but seamlessly integrated layers which vary in compositionand structure according to the distance from the surface. It istypically divided into four zones, superficial, middle (transitional),deep (radial) and calcified cartilage. The superficial zone is a thindense layer which forms the gliding surface of the joint and providessupport and protection. It is composed of thin collagen fibrils alignedparallel to the joint surface, with elongated inactive chondrocytes alsoaligned with the surface. The middle zone is thicker than thesuperficial zone and contains spherical cells and larger collagenfibrils that are orientated in a more random fashion. In the deep zonethe cells are spherical and are arranged in columnar orientation.Collagen fibrils in this zone are arranged perpendicular to the surfaceand insert into the calcified cartilage zone providing a transition andmechanical fixation between cartilage and bone (Newman A P.; 1998).Beneath the calcified cartilage zone lies the subchondral bone.Cartilage has a poor ability to regenerate itself due to the sparsedistribution and low mitotic activity (differentiation) of the articularchondrocytes and the avascular nature of the tissue.

Superficial damage to the articular cartilage almost inevitably leads tothe development of osteoarthritis (OA) within a joint. Cartilage damageand osteoarthritis affect at least 40 million Americans alone per annum,with an associated cost of approximately $105 billion. OA is estimatedto be the fourth leading cause of disability by 2020; affecting 9.6% ofmen and 18% of women aged over 60 years in Europe (Marketdevices/drivers; Mintel April 2007). Currently used surgical repairtechniques fall into three categories: 1) osteochondral grafting, 2)bone marrow stimulation techniques and 3) Autologous ChondrocyteImplantation (ACI). Osteochondral grafting or mosaicplasty involves theremoval of cylindrical osteochondral pieces from non-weight bearingareas of the articular cartilage and subsequent transfer of thesecylindrical plugs into debrided full thickness defects. Osteochondralgrafts can be autologous or allologous, depending on the defect size(allografts being used for larger defects). The major disadvantage ofautografts is the risk of donor site morbidity. The disadvantages ofallografts include the risk of disease transmission and tissuerejection.

Bone marrow stimulation techniques include abrasive chondroplasty,Pridie drilling and the microfracture technique. These techniques areall aimed at surgically creating access to the bone marrow, allowingblood flow into the defect site which in turn induces spontaneous repair(Beris A E; 2005). This forms a blood clot which traps proteins, lipids,red and white blood cells, platelets, growth factors and blood bornecells (Hunziker E B, 2001). Spontaneous repair occurs in the area,usually consisting of the production of fibrocartilaginous tissue (BerisA E; 2005). In the Pridie drilling technique, 2.0-2.5 mm diameter holesare drilled into the subchondral bone marrow space in areas beneath thelesion. The microfracture technique is a modification of Pridiedrilling, the only difference being that the holes drilled areconsiderably smaller (approximately 0.5 to 1.0 mm in diameter). Thesuccess with these surgical techniques has been limited as thefibrocartilage repair tissue which forms has poor mechanical properties,does not perform as well as hyaline cartilage and degenerates over time.

The Autologous Chondrocyte Implantation (ACI) technique involvesimplantation of chondrocytes into the defect site. It is a two stageprocess; the first step involves harvesting healthy articular cartilagesegments in order to obtain chondrocytes. These chondrocytes arecultured in vitro until sufficient numbers have been produced forimplantation into the defect. The second step involves clearing thelesion to reveal healthy cartilage and subchondral bone. A piece ofperiosteum is then sutured across the lesion and cultured chondrocytesare inserted into the defect, beneath this periosteal layer. The cellsthen attach to the defect walls and produce extra-cellular matrix. Thedisadvantages of this technique include poor retention of the implantedcells within the defect site and phenotypic transformation anddedifferentiation of chondrocytes during expansion in vitro. The defectsite is also incapable of load bearing and requires protection for theduration of the recovery period which may be several months.

More recently, membranes and scaffolds have been developed for therepair of cartilage tissue, both alone and in combination with growthfactors and cells. One example of the use of scaffolds in cartilagerepair is Matrix-induced Autologous Chondrocyte Implantation (MACI,Genzyme, The Netherlands) which involves the seeding of cells in amembrane prior to implantation resulting in better retention of cells inthe defect site. Other commercial examples of scaffolds used forcartilage repair include Chondro-Gide, (Geistlich), CaReS®—CartilageRepair System (Arthro Kinetics), Neocyte (Advanced Tissue Sciences(ATS), Atelocollagen (Koken), Menaflex (ReGen Biologics), andChondromimetic (Orthomimetics). A number of research groups arecurrently developing alternative cartilage repair scaffolds usingvarious materials; including polyglycolic acid (PGA), polylactic acid(PLA), collagen, gelatin and fibrin, and various scaffold productiontechniques; including freeze-drying, solid free-form fabrication,compaction and gelation.

The need for layered scaffolds for osteochondral defect repair has alsobeen identified and as a result, layered scaffold constructs arebeginning to emerge. Layered methods that have been used includesuturing (WO 96/024310) or gluing (US 2006/0083729 A1) the layerstogether. Disadvantages with these techniques include the introductionof an additional material into the defect site, (i.e. the suturematerial or adhesive) and also the possibility of voids being present atthe interface between the two materials, leading to lower interfacialadhesion strength and reduced cellular infiltration.

Solid-free form fabrication techniques have also been used to producelayered scaffolds (Sherwood J K, et al.; 2002). Other layered scaffoldshave been produced using a combination of a base ceramic scaffoldproduced though compaction and sintering and polymer scaffold top layersproduced through freeze-drying or gelation of the top scaffold layer(U.S. Pat. No. 0,113,951).

Tampieri et al. (Design of graded biomimetic osteochondral compositescaffolds, Biomaterials, vol 29, no 26, September 2008) describes a3-layered scaffold, with each layer containing varying amounts ofcollagen, hyaluronic acid, hydroxyapatite and magnesium-hydroxyapatite.The individual layers were producing by combining the various componentsto form gels, which were then crosslinked. The layered structure wasproduced using a knitting procedure. The multi-layer construct was thenfreeze-dried using a freezing temperature of −25° C. The method used inTampieri to produce the cartilaginous upper layer involves adding NaOHto a 1 wt % type 1 collagen suspension to form a gel. Hyaluronic acidwas added to the gel. The intermediate bony layer (tidemark) and lowerbony layer were produced by adding different quantities of H₃PO₄ andCa(OH)₂ to allow the formation of hydroxyapatite through a directnucleation process. The gels were cross-linked using the cross-linkingagent 1,4 butanediol diglycidyl ether (BDDGE). The layers were thenpiled up and a knitting procedure was used to avoid delamination of thelayers at the interface. The use of a knitting procedure isdisadvantageous as it requires the use of addition non-collagenousmaterials, such as PGA or PLA fibres, which have an effect on thebiocompatibility of the scaffold. Using a knitting procedure alsodamages the pore structure of the scaffold and results in seams or areasof heterogeneous lamination at the interface between layers. As a resultcellular infiltration through the scaffold is restricted. Additionally,freeze-drying of the entire scaffold is carried out in one step and as aresult the structure of the individual layers cannot be separatelycontrolled.

STATEMENTS OF INVENTION

The invention relates to a multilayer scaffold suitable for use intissue-engineering applications, for example, osteochondral defectrepair, tendon and ligament repair, vascular repair, tracheal defectrepair, and skin repair. The multilayer scaffold of the invention in itssimplest form comprises two layers, but may comprise three, four or morelayers. The scaffold is made using a process which employs an iterativefreezing technique, whereby each layer is subjected to an individualfreezing step. Each step may be simply freezing, or freezing followed bysublimation under vacuum (hereafter “lyophilisation” or“freeze-drying”). In each case, the freezing step ideally employs acontrolled constant cooling rate freezing method The purpose of thisiterative freezing technique is to allow fabrication of each layer usingconditions which are independent of each other which, in turn, allowseach layer to have different characteristics (for example, differentpore morphology and pore architecture). The iterative freezing techniqueof the process of the invention also provides a multi-layer scaffold inwhich the distinct layers are seamlessly integrated—that is to say, thepore structure is continuous across the different layers. When freezingalone is employed to produce a layer in the scaffold, the next layer maysimply be poured onto the formed layer in the form of a slurry. However,when lyophilisation is employed to form a layer, the lyophilised layeris typically re-hydrated prior to the next layer being poured. The finalstep typically involves lyophilisation of the multi-layer structure toprovide the formed scaffold. As an example, a process of the inventionfor forming a two-layer scaffold involves forming a first layer byfreezing or lyophilisation, pouring a second slurry onto the firstlayer, and lyophilising the two layers to produce a two-layer scaffold,wherein when the first layer is formed by lyophilisation (as opposed tofreezing alone) and is re-hydrated prior to the second layer beingformed. Fabrication of such a 2-layer construct is described inembodiment 1. This process can then be repeated to produce furtherlayers. The fabrication of a 3-layer scaffold in which the compositionsof the constituent layers of the structure are designed to closelyreplicate the morphology and composition of anatomical osteochondraltissue is described in embodiment 2.

According to the invention, there is provided a method for producing amulti-layer collagen scaffold comprising the steps of:

preparing a first suspension of collagen and freezing or lyophilisingthe suspension to provide a first layer;

-   -   optionally preparing a further suspension of collagen and        pouring the further suspension onto the layer formed in the        previous step to form a further layer, and freezing or        lyophilising the layers, wherein when the layer formed in the        previous step is formed by lyophilisation the lyophilised layer        is re-hydrated prior;    -   optionally, repeating step B to form one or more further layers;        and    -   preparing a final suspension of collagen and pouring the final        homogenous suspension onto the uppermost layer to form a final        layer, and freeze-drying the layers to form the multi-layer        collagen composite scaffold.

The process of the invention provides a multi-layer collagen scaffold inwhich each layer is formed in a separate freezing or lyophilisationstep. As such, the freezing or lyophilisation conditions of each stepmay be independently varied which allows the pore structure of eachlayer of the layered scaffold to be individually optimised. This is ofvital importance as chondrocytes and osteoblasts require vastlydifferent conditions for optimal in vivo behaviour (Engler et al.,2006). This is distinct from the process described in Tampieri et al. inwhich each layer is formed independently as a gel, the different layersare physically sutured together, and all layers are lyophilised in asingle step using the same lyophilisation conditions. Thus, while themethod described by Tampieri et al. allows for the production of ascaffold having layers which are compositionally distinct, the fact thatall layers are lyophilised together means that the pore architecture ineach layer cannot be independently controlled or varied. Additionally,the step of suturing layers together followed by a single lyophilisationstep results in large voids being formed at the interface betweenlayers, and a discontinuous pore architecture across the scaffold (seeFIGS. 1 and 8 in Tampieri et al.). This is in contrast to the scaffoldsmade using the process of the invention in which the pore architectureacross the layers is continuous and seamless, and uninterrupted by seamsor large voids (See FIG. 9 below). In the present invention the scaffoldis produced using an iterative layering technique, where the layers areeither frozen or freeze-dried and rehydrated prior to addition of thefollowing layer.

Ideally, one more or all of the suspensions of collagen are homogenoussuspensions.

Suitably, each layer is typically compositionally distinct.

Thus, the invention relates to a process for producing a multi-layerscaffold in which each layer typically has a porous structure, theprocess comprising an iterative layering technique in which each layeris formed by freezing, or lyophilisation followed by re-hydration, priorto addition of a following layer. Suitably, the scaffold is collagenbased, and each layer is formed from a suspension of collagen in asolvent, typically a weak acid solvent, suitably a homogenous suspensionof collagen, which is initially in the form of a slurry, wherein thelayer is formed (i.e. solidified) by freezing or lyophilisation. When alayer is lyophilised, the layer is typically re-hydrated using the samesolvent as used in the collagen suspension, ideally a weakly acidicsolvent.

In one preferred embodiment of the invention, at least two of the layersin the scaffold are formed by lyophilisation. In a preferred embodimentof the invention, all layers in the scaffold are formed bylyophilisation.

In one embodiment, the scaffold comprises two layers. In this case,steps B and C are omitted. Preferably, the scaffold has three layers, inwhich case step B is carried out once.

Thus, the process of the invention is an iterative process in which afirst layer is formed by freezing or freeze-drying, then a subsequentlayer is poured onto the first layer and the composite is frozen orfreeze-dried, etc. A process in which layers are formed by freezing orfreeze-drying and, after separate formation, the formed layers areadhered together by, for example gluing or suturing, is typicallyexcluded. Where the process involves forming the layers by freezing, thefinal layer will be formed by freeze-drying. Where the process involvesforming each layer by freeze-drying, the formed layer or layers arere-hydrated prior to pouring the next layer.

The term “homogenous suspension” should be understood to mean asuspension of collagen in a solvent (for example a weak acid) in whichthe collagen is homogenously distributed throughout the solvent.Techniques for providing a homogenous suspension of collagen aredescribed below, and will be known to those skilled in the art. Ideally,the homogenous suspensions of collagen are provided in a slurry form.The suspension(s) comprise collagen, and optionally one or moreadditional suitable components selected from one, two or more of: amineral phase component such as a calcium phosphate (i.e. ahydroxyapatite); a polymer, preferably a biological polymer, for examplepoly(lactic-co-glycolic acid) (PLGA) or alginate or a glycosaminoglycan(GAG), such as chondroitin sulphate or hyaluronic acid, or a combinationof GAGs; and a biologic. The term “biologic” should be understood tomean a biologically-active molecule—examples of such molecules includenucleic acids, for example genes, DNA, RNA, low molecular weight nucleicacids, proteins, polypeptides, and peptides, hormones, growth factors,cytokines, metabolites and cells. The homogenous suspension of collagencomprises collagen homogenously distributed throughout the suspension.Typically, the collagen (and other components such as hydroxyapatitewhen included) is suspended in an acid solution. The molarity of theacid solution employed for making each layer may vary. Thus, in oneembodiment, the molarity of the acid solution used for making the bottomlayer may be 0.5M, whereas the molarity of the solution employed inmaking the intermediate and top layers may 0.05M In cases where theprocess involves re-hydrating a formed layer or layers, the layer(s) arere-hydrated in an acidic solution, generally a similar or identicalacidic solution to that used in forming the layer.

In one embodiment, the scaffold (or each layer in the scaffold) has aporosity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98%.Ideally, the scaffold has a porosity of at least 98%, ideally at least98.5%. A method of determining % porosity is described below.

In one embodiment, the scaffold (or each layer in the scaffold) has anaverage pore diameter of at least 80, 85, 90, 95, 96, 97, 98, 99 or 100microns. A method of determining average pore diameter is describedbelow.

Typically, each layer in the scaffold is compositionally distinct. Inthis specification, the term “compositionally distinct” should be takento mean that the layers differ in terms of their composition and/ormorphology. In a preferred embodiment, the layers differ in terms of aparameter selected from the group consisting of: hydroxyapatite content;type of collagen; amount of collagen; type of GAG; and quantity of GAG.In one embodiment, each layer comprises one or more constituentsselected from the group consisting of: Type I Collagen; a non-Type ICollagen (for example Type II Collagen); and a mineral phase (forexample a hydroxyapatite). Ideally, in an embodiment in which thescaffold comprises three layers, the first layer comprises collagen andhydroxyapatite, the second layer comprises collagen and hydroxyapatitein which the collagen content is different from the first layer, and thethird layer comprises collagen and little or no hydroxyapatite, andoptionally a polymer and/or a biologic.

In one embodiment of the invention, the method is a method of producinga three-layer collagen scaffold that typically mimics both themorphology and composition of healthy anatomical osteochondral tissue,and in which a first outer layer comprises collagen, typically Type Icollagen, and hydroxyapatite. This layer mimics the subchondral bone.Suitably, the inner layer comprises a type I collagen, non-Type ICollagen (ideally Type II collagen), hydroxyapatite and, optionally, oneor more GAGs. This layer mimics the intermediate articular calcifiedcartilage. Typically, the second outer layer comprises a composite ofType I collagen, non-Type I collagen (ideally Type II collagen) andoptionally one or more GAGs and/or a biologic. This layer mimics theoverlying cartilaginous layer.

Where the scaffold is a three-layer scaffold, the method typicallycomprises the steps of:

-   -   preparing a first homogenous suspension of collagen and        lyophilising the suspension to provide a first layer;    -   rehydrating the formed first layer;    -   preparing a second homogenous suspension of collagen and pouring        the second homogenous suspension onto the re-hydrated first        layer to form a two-layered composite, and lyophilising the        two-layer composite;    -   re-hydrating the two-layer composite; and    -   preparing a third homogenous suspension of collagen and pouring        the third homogenous suspension onto the two-layer composite to        form a three-layer composite, and lyophilising the three-layer        composite to form the three-layer collagen scaffold.

It will be clear that the process of the invention may be employed toproduce multi-layer collagen scaffolds having two, three, four or morelayers. Additionally, it will be clear that the process is an iterativeprocess for forming a polyphasic layered scaffold in which layers aresequentially added to the composite by a process of freezing orlyophilising, and in which after pouring of a new layer, the compositelayered structure is frozen or lyophilised.

Ideally, the multi-layer collagen composite scaffold is cross-linked.Typically, the composite scaffold is cross-linked by one or more of themeans selected from the group comprising: dehydrothermal (DHT)cross-linking; and chemical cross-linking. Suitable chemicalcross-linking agents and methods will be well known to those skilled inthe art and include 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride (EDAC) or Glutaraldehyde. Ideally, the scaffold iscross-linked using DHT and EDAC cross-linking. Cross-linking can becarried out at any stage of the fabrication process. In a preferredembodiment, scaffold pore symmetry can be controlled by varying thedegree of cross-linking within each respective layer using cross linkingmethods familiar to one skilled in the art. Similarly, in anotherembodiment, scaffold permeability or flow conductivity can be varied byvarying the mechanical properties of the scaffold using either crosslinking or other stiffness improvement methodologies known to oneskilled in the art.

Typically, the first homogenous suspension comprises collagen, ideallyType I collagen, and a mineral phase, ideally hydroxyapatite (referredto herein as “bottom” or “bone” layer). Ideally, the second homogenoussuspension comprises collagen, typically two types of collagen such asType I and a non-Type I collagen (typically Type II collagen), and amineral phase, ideally hydroxyapatite I (referred to herein as“intermediate” layer). Suitably, the third homogenous suspensioncomprises collagen, typically two types of collagen such as Type I and adifferent collagen such as Type II collagen, and optionally a polymerfor example a GAG, such as chondroitin sulphate or hyaluronic acid or acombination of GAGs (referred to herein as “top” or “cartilage” layer).Typically, the pore size of the top layer is greater than that of theintermediate layer. Suitably, the pore size of the intermediate layer isgreater than that of the bottom layer. The ratio of Type I collagen toType II collagen in the top (cartilage) layer can vary from 1:0 to 0:1.Ideally, it varies from 1:4 to 4:1 (w/w).

In a preferred embodiment, the pore size and/or pore size distributiongradient are varied within each layer independently. This can beachieved for example, by varying the slurry thickness used for eachlayer. Additionally, the individual layer thickness can be varied ineach of the layers of the multilayer scaffold between 1 mm and 15 mmusing different volumes of slurry during the freezing or lyophilisationprocess. The shape of the scaffold produced can also be varied by usingcontoured trays which are tailored to the required anatomical curvature.

Thus, in a particularly preferred embodiment of the invention, theprocess comprises the steps of:

-   -   preparing a first homogenous suspension of Type I collagen and        HA in the form of a slurry and freezing or lyophilising the        slurry to provide a first layer;    -   when the first layer is formed by freeze-drying, rehydrating the        formed first layer;    -   preparing a second homogenous suspension of Type I collagen, a        non-Type I collagen (i.e. Type II collagen), HA and optionally        GAG in the form of a slurry and pouring the slurry onto the        first layer to form a two-layer composite, and freezing or        lyophilising the two-layer composite;    -   when the second layer is formed by freeze-drying, re-hydrating        the second layer;    -   preparing a third homogenous suspension of Type I collagen and a        non-Type I collagen (i.e. Type II collagen) and optionally a        polymer (for example one or more GAG's) and/or a biologic and        pouring the third homogenous suspension onto the two-layer        composite to form a three-layer composite, and lyophilising the        three-layer composite to form the three-layer collagen scaffold;        and    -   optionally, cross-linking one or more of the layers of the        scaffold.

Ideally, all layers of the scaffold are cross-linked in a singlecross-linking step. However, each layer may be cross-linked in separatesteps, for example by cross-linking a layer or layers following afreezing or lyophilisation step.

The process of the invention typically involves lyophilising the layers,either after each iterative step, and/or as part of the final step. Thisis a process in which the slurry is frozen, typically to a finalfreezing temperature of from −10° C. to −70° C. and then sublimatedunder pressure. In one embodiment, the desired final freezingtemperature is between −10° C. and −70° C. Suitably, the desired finalfreezing temperature is between −30° C. and −50° C. Typically, thedesired final freezing temperature is between −35° C. and −45° C.,ideally about −40° C.

In one embodiment of the invention, freezing or freeze-drying is carriedout at a constant cooling rate. This means that the rate of cooling doesnot vary by more than +/−10% of the target cooling rate, i.e. if thedesired rate of cooling is 1.0° C./min, and the actual rate of coolingvaried between 0.9° C./min and 1.1° C./min, this would nonetheless stillbe considered to be a constant cooling rate. Typically, the constantcooling rate is between 0.1° C./min to 10° C./min. Preferably,freeze-drying is carried out at a constant cooling rate of between 0.5°C./min to 1.5° C./min. More preferably, freezing or freeze-drying iscarried out at a constant cooling rate of between 0.8° C./min to 1.1°C./min. Typically, freezing or freeze-drying is carried at a constantcooling rate of about 0.9° C./min. The temperature of the freeze-dryingchamber at a start of the freeze-drying process (i.e. when the slurry isplaced in the chamber) is usually greater than 0° C., preferably atabout ambient temperature.

The sublimation step is generally carried out after the final freezingtemperature is reached. This step involves heating the freeze-dryingchamber to a sublimation temperature (generally about 0° C.), preferablyat a constant heating rate. The process typically includes a finalsublimation step where an ice phase in the formed scaffold is sublimatedunder vacuum for a suitable period of time.

In another embodiment of the invention, the freeze-drying processcomprises an annealing step. Typically, this step involves increasingthe temperature in the freeze-drying chamber after the final freezingtemperature has been reached, and typically holding the increasedtemperature for a period of time before initiating the drying stage. Forexample, if the final freezing temperature is −20° C., the annealingstep may be carried out by ramping up the temperature to −10° C., andholding at that temperature for a time sufficient to allow existing icecrystals grow, before finally drying the scaffold. The annealing timemay be varied according to the pore characteristics desired; however,annealing times of between 15 minutes and 120 hours are preferred.

Generally, the HA employed in the present invention is in powder form.Suitably, the HA powder is selected from the group comprising: sinteredHA powder; and unsintered HA powder. Examples of suitable sintered, andunsintered, HA powders suitable for the present invention will be knownto the person skilled in the art, and are provided below. It will beappreciated that the HA is employed as a mineral phase in the layerswhere it is employed. In this regard, it will be apparent to the skilledperson that the process and products of the invention may be embodied byreplacing HA with an alternative mineral phase such as, for example,brushite, α-TCP or β-TCP. Other suitable mineral phase materials will bewell known to the skilled person.

Typically, the HA powder has a particle size of between 10 nm and 100μm.

Suitably, the collagen employed in the present invention comprisescollagen fibres. Preferably, the collagen fibres comprise microfibrillarcollagen, preferably microfibrillar bovine tendon collagen for type Icollagen and porcine cartilage for type II collagen.

The homogenous suspension(s) of collagen comprises collagen suspended inan acidic solution. Typically, the acidic solution has a molarity of atleast 0.01M, Suitably, the molarity of the acidic solution is at least0.06M, 0.07M, 0.08M, 0.09M, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M or 1M. Ideally,the molarity of the acidic solution is between 0.4M and 0.6M. Ideallythe acid is an organic acid, preferably acetic acid, although otherorganic acids may be employed.

The lyophilised layer(s) is rehydrated in an acidic solution having amolarity of preferably at least 0.015M, preferably at least 0.02M, andtypically in a range of 0.02M to 0.03M, suitably about 0.023M to 0.027M,and ideally about 0.025M Ideally the acid is acetic acid, although otherweak organic acids may be employed.

In an embodiment of the invention in which the homogenous suspensioncomprises collagen and hydroxyapatite (CHA) slurry, the ratio of HA tocollagen in the suspension is typically greater than 1:10 (w/w), and themolarity of the acidic solution is greater than 0.01M. Typically, theratio of HA to collagen in the suspension is at least 2:10 (w/w), 3:10(w/w), 4:10 (w/w), 5:10 (w/w). In one preferred embodiment of theinvention the ratio of HA to collagen is from 1:10 (w/w) to 100:10(w/w), suitably from 1:10 (w/w) to 50:10 (w/w), suitably from 5:10 (w/w)to 30:10 (w/w).

In one preferred embodiment of the invention, the ratio of HA tocollagen in the first homogenous suspension is at least 5:10 (w/w), andwherein the molarity of the acidic solution is at least 0.1M. Typically,the molarity of the acidic solution is at least 0.50M.

In a preferred embodiment of the invention, the ratio of HA to collagenin the first homogenous suspension is at least 1:2 (w/w), 1:1 (w/w), 2:1(w/w), 3:1 (w/w), 4:1, or 5:1 (w/w). In one embodiment of the invention,the ratio of HA to collagen in the first homogenous suspension isgreater than 5:1 (w/w). Generally, when such levels of HA are employedin the suspension, the molarity of the acidic solution will be at least0.5M.

In a preferred embodiment, the amount of collagen in the suspension canvary from 0.5 g/L up to 50 g/L of acid solution ( 1/10 and 10 timesstandard collagen concentration respectively). Suitably, the amount ofcollagen in the suspension is between 1.0 g/L and 10.0 g/L, preferablybetween 3.0 g/L and 8.0 g/L, and more preferably between 4.0 g/L and 6.0g/L.

Typically, the acidic solution comprises an acetic acid solution.However, other organic acids may be employed to form the acidicsolution.

Suitably, the homogenous suspensions of collagen are formed inconditions suitable for minimising gelatinisation of the collagen. Onemethod of ensuring minimal gelatinisation of collagen during theproduction of the homogenous suspension is to maintain the suspension ata sufficiently low temperature, generally between 1° and 5° C., suitablyabout 4° C.

More recent advances in cartilage tissue engineering involve the use ofscaffolds as growth factor or gene carrier systems. There are a numberof essential growth factors providing regulatory effects on chondrocytesor stem cells involved in chondrocyte maturation and cartilageformation. These include the TGF-β superfamily, IFG, FGF, BMP, PDGF andEGF (Lee S H, Shin H; 2007). Miljkovic et al. (Miljkovic et al.; 2008)report on the successful delivery of BMP-4 for the treatment ofcartilage defects. Thus, in one embodiment of the invention, the processincludes an additional step of incorporating a biologic into themulti-layer collagen-composite scaffold. This could be achieved by, forexample, soaking the prepared scaffold in a solution containing thegrowth factor (or cells) of interest, through cross-linking or usingtranscription. Suitably, the biological material (biologic) is selectedfrom the groups of: cells; and biological growth factors. Typically, thebiological growth factors are selected from the group consisting of oneor more of the TGF-β superfamily, (IFG, FGF, BMP, PDGF, EGF) orcannabinoids. These growth factors can also be included during theproduction process as opposed to post-fabrication soaking of thescaffolds. Typically, the cells are selected from the group consistingof chondrocytes, osteoblasts or mesenchymal stem cells, although othercells may be employed. The invention also relates to a cell-seededtissue engineering construct comprising a multilayer collagen scaffoldaccording to the invention having cells incorporated into the scaffold,ideally into the pores of the scaffold. Cell-seeded tissue engineeringconstructs of the invention may be made by seeding a scaffold of theinvention with cells, and then culturing the cells in-vitro, prior touse (implantation) of the construct. Accordingly, the invention alsorelates to a method of producing a cell seeded tissue engineeringconstruct of the type comprising a multilayer collagen scaffoldaccording to the invention, wherein the cells are disposed within thepores of the scaffold, the method comprising the steps of seeding cellsfrom a host onto the scaffold, and culturing the cells on the scaffoldprior to implantation into a defect.

Additionally, these scaffolds are ideally suited for use as deliverymechanisms for gene therapy delivery, either through viral or non-viraldelivery vectors. The idea of a gene delivery vector contained within abiodegradable scaffold, although not new, is a recent development in thefield of regenerative medicine and the system has been coined as a ‘geneactivated matrix’ (GAM). Gene therapy can be a valuable tool to avoidthe limitations of local delivery of growth factors, including shorthalf-life, large dose requirement, high cost, need for repeatedapplications, and poor distribution.

The invention also relates to a multi-layer collagen scaffold obtainableby the process of the invention.

The invention also relates to the use of the multi-layer collagenscaffold obtainable by the process of the invention in repairingosteochondral defects, tendons and ligaments, vascular tissue, trachealtissue, or skin.

The invention also relates to a multi-layer collagen scaffold comprisinga plurality of freeze-dried layers in which a first layer comprisescollagen, typically Type I collagen, and HA, a second layer comprisesone or more types of collagen (typically Type I collagen and a non-TypeI collagen such as Type II collagen), HA and optionally one or moretypes of GAG, and a third layer comprises one or more types of collagen(typically two types of collagen, i.e. Type I and a non-Type I collagensuch as Type II collagen) and optionally one or more types of GAG andoptionally a biologic. Ideally, the layers in the scaffold are adheredtogether. In a preferred embodiment, the layers are freeze-driedtogether. However, other methods for adhering the layers together willbe apparent to the skilled person including, for example, suturing andadhesive. Preferably, the scaffold comprises a continuous porearchitecture extending across the layers of the scaffold.

The invention also relates to a multi-layer collagen scaffold comprisinga plurality of freeze-dried layers in which a first layer consistsessentially of collagen and HA, a second layer consists essentially of acollagen, HA and a GAG, and a third layer consisting essentially ofcollagen and GAG, wherein a ratio of HA in the first layer to HA in thesecond layer is at least 1:1 (w/w). Typically, the ratio of HA in thefirst layer to HA in the second layer is at least 3:1 (w/w), preferablyat least 4:1 (w/w), and ideally at least 5:1 (w/w). Suitably, thecollagen component in the first layer comprises (or consists essentiallyof) a single type of collagen, typically Type II collagen. Generally,the collagen component of the second and third layers comprises (orconsists essentially of) two types of collagen, suitably Type II and anon-Type I collagen.

The individual layer thickness can be varied in each of the layers ofthe multilayer scaffold between 1 mm and 15 mm using different volumesof slurry during the freezing or lyophilisation process. In a preferredembodiment, the pore size and pore size distribution gradient can bevaried within each layer independently by varying the volume of slurryused to produce each layer. The shape of the scaffold produced can alsobe varied by using contoured trays which are tailored to the requiredanatomical curvature.

Thus, the invention also relates to a multi-layer collagen-compositescaffold suitable for use in tissue and bone defect repair applicationsor tissue engineering applications, especially osteochondral defectrepair, and comprising a layered structure comprising at least threeporous scaffold layers that differ in at least a parameter selected fromcollagen content, collagen type, and hydroxyapatite content, wherein thescaffold has a continuous, and optionally variable, pore architectureextending across the scaffold (i.e. extending across the layers). Theterm “continuous pore architecture” means that the porous architectureextends across three layers without being interrupted by seams formed atthe interface between layers. Such a continuous (also referred to hereinas “seamless”) pore architecture can be clearly seen in FIG. 9 below,which shows a three-layer scaffold of the invention in which the porestructure of one layer is integrated into the pore structure of anadjacent layer without being interrupted by seams or areas oflamination. This can be contrasted with the pore structure of scaffoldshown in FIG. 8 of Tampieri et al, which is clearly discontinuous andincludes seams located at the interface between the 70/30 and 40/60layers. The term “variable pore architecture” means that the porearchitecture may also be variable across the layers (for example, thepore size may vary). In one embodiment, the porous structure of eachlayer is different, for example the formed scaffold may have a pore sizegradient extending across the layers due to each layer having differentpore size characteristics.

The invention also relates to a multi-layer collagen scaffold comprisinga plurality of porous freeze-dried layers in which the scaffold has acontinuous pore architecture extending across the layers, and in whichthe pore architecture of at least two of the layers is typicallydifferent. Ideally, the scaffold comprises a pore architecture gradientextending across the layers. This means that one or more porearchitecture characteristic, for example, pore size, pore sizehomogeneity or pore size distribution, is varied across the layers in agraded manner (for example, the pore size may increase from one layer tothe next, or the pore size homogeneity may increase from one layer tothe next). The pore architecture can also be varied in a manner thatwill result in a small pore size in the central layer and larger poresizes in the outer layers, or vice versa.

The invention also relates to a multi-layer collagen scaffold comprisinga plurality of porous freeze-dried layers in which the interface betweeneach layer is seamless, and in which the pore architecture of at leasttwo of the layers is typically different.

Suitably, the multi-layer collagen-composite scaffold is provided in theform of a core.

The invention also relates to a seamlessly integrated multilayerscaffold formed in a process that employs an iterative layering processthat allows independent control of (a) cooling rate (b) final freezingtemperature, (c) freezing gradient experienced by each layer.

The invention also relates to an integrated multilayer scaffold withcontinuous physical integration at the interface of each adjacent layer,wherein each layer comprises a pore architecture characteristic that isdifferent to the other layers. The pore architecture characteristic isfor example selected from pore size, pore size homogeneity and pore sizedistribution.

A highly porous multilayer/multi-region scaffold with distinct butphysically integrated regions, independently controlled pore size andpore architecture in each region of the scaffold, with a regionoptimised to produce maximum cartilage production, a region optimised toproduce maximum bone production and an intermediate region optimised toproduce calcified cartilage in between.

An integrated multilayer scaffold with distinct regions that havecontinuous physical integration across the layer interfaces/interfacialregions, in which each layer has at least one characteristic selectedfrom pore size, pore homogeneity, pore size distribution, compositionand mechanical properties that is different from the other layers.

A multilayer/multi-region scaffold with a continuous physical interfacebetween adjacent layers which allows a high degree of cellularinfiltration across all regions of the scaffold and which has theoptimal composition, pore architecture, porosity and mechanicalproperties to induce the differentiation of mesenchymal stem cells(MSCs) into chondrocytes in the cartilage region of the scaffold andinto osteoblasts in the bone region of the scaffold.

A multilayered, integrated scaffold replicating the anatomicalcomposition and structure of native osteochondral tissue for use in therepair of osteochondral tissue.

The term “integrated” as used above should be understood to mean thatthe pore architecture of adjacent layers is continuous and notinterrupted by seams or voids.

A highly porous scaffold with a functionally graded structure, withvarying composition, pore size, pore homogeneity, permeability andmechanical properties, optimised for the repair of an osteochondraldefect.

The invention also relates to a method of repairing a tissue defect suchas an osteochondral defect in a mammal, comprising the steps ofproviding a multi-layer collagen scaffold of the invention, shaping thescaffold to fit into the defect, optionally soaking the scaffold, eitherbefore or after shaping, in a solution of biological material, andinserting the scaffold into the defect. Optionally, the biologicalmaterial can be incorporated into the scaffolds during the fabricationprocess. Typically, the scaffold has a base layer comprising collagenand HA, and wherein the scaffold is inserted into the defect such thatthe base layer abuts the deepest part of the defect. In one embodiment,the defect is also shaped to ensure a good fit between the defect andthe scaffold. Examples of other tissue defects that may be repairedusing the scaffolds of the invention include mandibular/maxillofacialdefects, cardiovascular defects, tracheal reconstruction, cartilagedefect repair within any articulating joint within the skeletal system(e.g. hip, knee, shoulder, ankle, hand, foot, neck, spine), any softtissue defect within the body, rheumatoid arthritis, osteoarthritis, anyform of arthritis resulting in cartilage damage, repair of collateraldamage at articulating joints due to trauma (e.g. Anterior CruciateLigament, torn rotator cuff, dislocated/broken ankle, meniscal repair),ankle joint repair and soft tissue reconstruction (maxillofacial, tissueaugmentation).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Toluidine blue stained 10 μm transverse section of the bottomCHA layer of a 2-layer scaffold, showing the presence of hydroxyapatite(HA) particles within the scaffold struts. This confirms that HA remainswithin the scaffold during the ‘iterative layering process’. (Bottomlayer CHA scaffold=200 wt % HA)

FIG. 2: Pore structure of the 2-layer 200 wt % CHA/Col1 Scaffold. Thescaffold pore structure was investigated by embedding the scaffold andthen sectioning it to a thickness of 10 μm using a microtome. The sliceswere mounted on a glass slides and the scaffold struts were stainedusing Toluidine blue stain. FIG. 3 shows a micrograph of histologicalsections of the 2 layer 200 wt % CHA (base layer)/Col1 (top layer)scaffold imaged using light microscopy. The micrographs indicate thatthe base 200 wt % layers retains its pore structure during the‘iterative freeze-drying’ process. The top layer also displays equiaxedpore morphology.

FIG. 3: XRD patterns for the pure HA powder in blue and the bottom layerCHA scaffold in black, with the characteristic peaks for HA in red.(Bottom layer CHA scaffold=200 wt % HA). This shows that HA issuccessfully incorporated into the scaffold and that its phase purity isunaffected by the process.

FIG. 4: Comparison of the mechanical properties of a standard collagenscaffold, the bottom or bone layer (Layer 3—containing type I collagenand 200 wt % HA), the intermediate layer (Layer 2—containing equalamounts of type I collagen, type II collagen and HA) and the top orcartilage layer (Layer 1—containing equal amounts of type I and type IIcollagen), and a 3-layer scaffold following DHT treatment at 105° C. for24 hours.

FIG. 5: Permeability of the individual scaffold layers compared to thatof a pure collagen scaffold. (Bottom layer CHA=200 wt % HA, Intermediatelayer=type I collagen in 0.05M acetic acid:type II collagen in 0.05Macetic acid:200 wt % HA in 0.5M acetic acid (1:1:1), Top layer=type Icollagen in 0.05M acetic acid:type II collagen in 0.05M acetic acid(1:1).

FIG. 6: Representative micrographs of the pore structure of each of thecomponent layers of the 3-layer scaffold produced in isolation showingthe homogeneous pore architecture. (Bottom layer CHA=200 wt % HA,Intermediate layer=type I collagen in 0.05M acetic acid:type II collagenin 0.05M acetic acid:200 wt % HA in 0.5M acetic acid (1:1:1), Toplayer=type I collagen in 0.05M acetic acid:type II collagen in 0.05Macetic acid (1:1)).

FIG. 7: Porosity of a standard collagen scaffold, each of the componentlayers of the 3-layer scaffold produced in isolation, and a 3-layerscaffold, showing the high porosity of all scaffold variants. (Bottomlayer CHA=200 wt % HA, Intermediate layer=type I collagen in 0.05Macetic acid:type II collagen in 0.05M acetic acid:200 wt % HA in 0.5Macetic acid (1:1:1), Top layer=type I collagen in 0.05M acetic acid:typeII collagen in 0.05M acetic acid (1:1)).

FIG. 8: Comparison of the pore diameters of each of the component layersof the 3-layer scaffold produced in isolation. The average porediameters were found to vary from 112 μm (intermediate layer scaffold)136 μm (bottom layer scaffold). (Bottom layer CHA=200 wt % HA,Intermediate layer=type I collagen in 0.05M acetic acid:type II collagenin 0.05M acetic acid:200 wt % HA in 0.5M acetic acid (1:1:1), Toplayer=type I collagen in 0.05M acetic acid:type II collagen in 0.05Macetic acid (1:1)).

FIG. 9: SEM micrographs of the 3-layer scaffold showing the highlyporous structure, high degree of pore interconnectivity and seamlessintegration of the component layers. (Bottom layer CHA=200 wt % HA,Intermediate layer=type I collagen in 0.05M acetic acid:type II collagenin 0.05M acetic acid:200 wt % HA in 0.5M acetic acid (1:1:1), Toplayer=type I collagen in 0.05M acetic acid:type II collagen in 0.05Macetic acid (1:1). This example was produced using the ‘iterativelayering technique’ and freeze-drying using a freezing temperature of−40° C.).

FIG. 10: Cell numbers for 3-layer scaffolds compared to collagenscaffolds at 7 and 14 days showing an increase in cell number ofapproximately 50% from day 7 to day 14 for both the collagen and the3-layer scaffolds. (Bottom layer CHA=200 wt % HA, Intermediatelayer=type I collagen in 0.05M acetic acid:type II collagen in 0.05Macetic acid:200 wt % HA in 0.5M acetic acid (1:1:1), Top layer=type Icollagen in 0.05M acetic acid:type II collagen in 0.05M acetic acid(1:1). This example was produced using the ‘iterative layeringtechnique’ and freeze-drying using a freezing temperature of −40° C.).

FIG. 11: Scaffold contraction of the 3-layer scaffold compared to astandard collagen scaffold at 7 and 14 days post seeding with MC3T3-E1mouse pre-osteoblast cells. The 3-layer scaffold was found to contractto a lesser extent than the standard collagen scaffold. (Bottom layerCHA=200 wt % HA, Intermediate layer=type I collagen in 0.05M aceticacid:type II collagen in 0.05M acetic acid:200 wt % HA in 0.5M aceticacid (1:1:1), Top layer=type I collagen in 0.05M acetic acid:type IIcollagen in 0.05M acetic acid (1:1). This example was produced using the‘iterative layering technique’ and freeze-drying using a freezingtemperature of −40° C.).

FIG. 12: Histologically prepared, haematoxylin and eosin (H&E) stainedtransverse sections of the 3-layered scaffold following 14 days in vitroculture with MC3T3-E1 mouse pre-osteoblast cells. (Bottom layer CHA=200wt % HA, Intermediate layer=type I collagen in 0.05M acetic acid:type IIcollagen in 0.05M acetic acid:200 wt % HA in 0.5M acetic acid (1:1:1),Top layer=type I collagen in 0.05M acetic acid:type II collagen in 0.05Macetic acid (1:1). This example was produced using the ‘iterativelayering technique’ and freeze-drying using a freezing temperature of−40° C.).

FIG. 13: Histological image of a type I collagen/hyaluronic (HyA) acidscaffold containing 10 mg/ml of HyA, following 21 days culture with ratMSCs in chondrogenic medium, stained with fast green, safranin-O andHaemotoxylin.

FIG. 14: Layer adhesion strength test results for the 2-layer and3-layer scaffolds. (2-layer scaffolds=Bottom layer CHA=200 wt % HA, Toplayer=type I collagen in 0.05M acetic acid; 3-layer scaffolds=Bottomlayer CHA=200 wt % HA, Intermediate layer=type I collagen in 0.05Macetic acid:type II collagen in 0.05M acetic acid:200 wt % HA in 0.5Macetic acid (1:1:1), Top layer=type I collagen in 0.05M acetic acid:typeII collagen in 0.05M acetic acid (1:1). Both produced using the‘iterative layering technique’ and freeze-drying using a freezingtemperature of −40° C.).

FIG. 15: 3-layered scaffold a) SEM image showing scaffold microstructureb) H&E stained sections at 14 days post seeding.

FIG. 16: A—ChondroColl—3 layered scaffold. B—Histological images ofnormal cartilage superficial, intermediate and deep zones showingorientation of chondrocytes.

FIG. 17: Histological images of normal cartilage superficial,intermediate and deep zones showing orientation of chondrocytes.

DETAILED DESCRIPTION OF THE INVENTION Materials and Methods Example 1

Embodiment 1 relates to the production of a two layer scaffold using the‘iterative freeze-drying’ technique. The invention consists of a basebone layer composed of type I collagen and preferably 200 wt %hydroxyapatite, but this can range from 0 wt % HA up to 500 wt % HA. Thetop cartilage layer is composed of type I and type II collagen. Theratio of type I collagen to type II collagen can be from 0:1 to 1:0.

The preferred 200 wt % collagen/HA (CHA) slurry for the bone layer ofthe scaffold is prepared as follows: 240 ml of preferably 0.5 M aceticacid (this can range from 0.05M to 10M) was prepared by adding 6.9 mlglacial acetic acid to 233.1 ml of distilled deionised water. 1.2 g ofmicrofibrillar bovine tendon collagen (Collagen Matrix Inc., NJ, USA)was placed into a beaker and 100 ml of 0.5 M acetic acid solution wasadded. The beaker was refrigerated at 4° C. overnight to allow thecollagen to swell and thus blend more easily. A WK1250 water coolingsystem (Lauda, Westbury, N.Y., USA) was used to cool a glass reactionvessel to 4° C. The collagen and acetic acid solution were added to thereaction vessel. 100 ml of the 0.5 M acetic acid solution was added tothe beaker to remove any remaining collagen and then poured into thereaction vessel. The components were blended using an IKA Ultra TurraxT18 overhead blender (IKA Works Inc., Wilington, N.C.) at 15,000 rpm for90 minutes. The slurry components were maintained at 4° C. duringblending to prevent denaturation of the collagen as a result of the heatgenerated during the process.

2.4 g hydroxyapatite (HA) powder (Captal ‘R’ Reactor Powder, PlasmaBiotal, UK) was added to 40 ml of the 0.5 M acetic acetic solution. TheHA acetic acid suspension was mixed in a syringe-type delivery devicecreating a homogenous suspension of HA particles within the acetic acidsolution. 10 ml of the HA suspension was added to the collagen slurryduring blending by placing the tip of the HA delivery device tube intothe vortex centre created by the blender. This ensured rapid dispersionof the HA suspension through the collagen slurry. 10 ml of the HAsuspension were added to the slurry every hour (4 additions of 10 ml ofHA suspension in total). The slurry was blended for a further 60 minutesfollowing addition of the final 10 ml of HA giving a total blend time of330 minutes (5½ hours). The interval between the addition of thealiquots of the HA suspension can be varied from 30 to 240 minutes. Thenumber of additions can also be varied, preferably HA is added in atleast 2 aliquots.

CHA slurries containing other quantities HA can be produced by varyingthe quantity of HA added, for example, for a 100 wt % CHA slurry, 1.2 gof HA powder would be added to 40 ml of 0.5M acetic acid. Examples ofsuch slurries are described in International Patent Application(Publication) No. WO2008/096334 (Royal College of Surgeons in Ireland).

Following blending, the slurry was degassed in a conical flask connectedto a vacuum pump for 30 minutes to remove unwanted air bubbles withinthe slurry. 15.6 ml of the slurry was then placed in a 60 mm×60 mmsquare 304 grade stainless steel tray. The slurry was then freeze-driedin a Virtis (VirTis Co., Gardiner, N.Y., USA) freeze-drier. Thefreeze-drying cycle used can be varied in order to produce scaffoldswith different pore structures. This is achieved by varying the freezingtemperature used from −10° C. to −70° C. The −40° C. freeze-drying cycleconsisted of the following steps: The tray was placed on a shelf in thefreeze drier at 20° C. The cycle involved cooling the shelf to −40° C.at a preferred constant rate of 0.9° C./min, based on the findings of aprevious study (O'Brien F J; 2004). The cooling rate selected can rangefrom 0.1° C./min to 10° C./min. The shelf temperature was then heldconstant for 60 minutes to complete the freezing process. The shelftemperature was then ramped up to 0° C. over 160 minutes. The ice phasewas then sublimated under a vacuum of 200 mTorr at 0° C. for at least 17hours to produce the porous CHA scaffold.

A type I collagen (Col1) slurry was produced as follows:

The preferred Col1 slurry contains 5 g/l of type I collagen suspended in0.05 M acetic acid. The quantity of Col1 can be varied between 5 g/l and50 g/l. The acetic acid concentration used can range between 0.01 M and10 M. 240 ml of 0.05 M acetic acid was prepared by adding 0.69 mlglacial acetic acid to 239.31 ml of distilled deionised water. 160 ml of0.05 M acetic acid was added to 0.8 g of Col1 and left to swellovernight in the refrigerator at 4° C. A WK1250 water cooling system(Lauda, Westbury, N.Y., USA) was used to cool a glass reaction vessel to4° C. The collagen and acetic acid solution was added to the reactionvessel and the components were blended for 90 minutes. Followingblending, the slurry was degassed in a conical flask connected to avacuum pump for 30 minutes to remove unwanted air bubbles within theslurry. The slurry was placed in a bottle and stored in a refrigeratorat 4° C. The acetic acid concentration used to produce the Col1 slurrycan be varied from 0.01M and 10M.

Iterative Layering Process

The bone layer CHA scaffold was rehydrated in an acetic acid solution inorder to prevent collapse of the scaffold following addition of thesecond layer slurry and also to prevent excessive infiltration of thesecond layer slurry into the base scaffold. The concentration of theacetic acid solution can be varied from 0.001M acetic acid to 5M, with0.025 M acetic acid solution being the preferred concentration. 800 mlof 0.025 M acetic acid was prepared by adding 1.1 ml glacial acetic acidto 798.9 ml of distilled deionised water. Rehydration involved fillingthe 60 mm×60 mm freeze-dying tray with acetic acid and placing thescaffold into it. This was then placed under vacuum until the scaffoldwas fully rehydrated and air bubbles had been removed from the scaffold.Excess acetic acid was removed using a pipette. 15.6 ml of the top layercollagen slurry was carefully pipetted on top. The two layer constructwas then returned to the freeze-dryer and freeze-dried using thefreeze-drying process described above.

Example 2

Embodiment 2 relates to a three layer scaffold, the base layer of thescaffold has similar structural and compositional properties to thesubchondral bone layer and consists of the primary constituents of bone;type I collagen (the organic phase) and hydroxyapatite (the mineralphase). The intermediate layer has a similar composition to calcifiedcartilage and consists of type II collagen which is present in cartilageand also type I collagen and hydroxyapatite (present in bone). The toplayer, modelled on the superficial to the deep zones of articularcartilage, comprises type I and type II collagen.

Bone Layer:

The bone layer consisted of a CHA scaffold, with the amount of HApresent varying between 0 wt % and 500 wt %. The CHA slurry wasfabricated and freeze-dried as described in embodiment 1 above.

Intermediate Layer:

The intermediate layer consisted of type I collagen (Col1) (CollagenMatrix Inc., NJ, USA), type II collagen (Col2) (Porcine type IIcollagen, Biom'up, Lyon, France) and hydroxyapatite (HA) (Captal ‘R’Reactor Powder, Plasma Biotal, UK).

A type I (Col1) slurry was produced as described in embodiment 1. Thetype II collagen (Col2) slurry can contain from 5 g/l to 50 g/l type IIcollagen. The 5 g/l Col2 slurry is produced by placing 0.2 g of Col2into a glass beaker and then adding 40 ml of acetic acid solution. Theacetic acid concentration used can be varied from 0.01 M to 0.5 M. Thesolution was refrigerated at 4° C. overnight to allow the collagen toswell. The solution was placed on ice and blended using a homogeniserfor 30 minutes to produce a homogenous slurry. The slurries containingthe greater quantities of Col2 are produced by increasing the amount ofCol2 added, for example a 1% Col2 slurry contains 0.4 g of Col2 in 40 mlof 0.05 M acetic acid.

The intermediate layer slurry was produced by combining the CHA slurry,Col1 slurry and Col2, produced as described in embodiment 1 and 2,slurry into a glass beaker. The 3 slurries were mixed by blending usinga homogeniser for 30 minutes until a homogenous solution was produced.The homogenous slurry was then degassed to remove air bubbles by placingthe beaker in a vacuum chamber connected to a vacuum pump. The amount ofeach component slurry in the intermediate layer can be varied between 0%and 100%.

Prior to addition of the intermediate layer slurry to the bone layerscaffold, the bone layer scaffold was rehydrated. This is necessary inorder to prevent scaffold collapse. The preferred rehydration medium wasa 0.025 M acetic acid solution. 800 ml of 0.025 M acetic acid wasprepared by adding 1.1 ml glacial acetic acid to 798.9 ml of distilleddeionised water.

A 60 mm×60 mm square 304 grade stainless steel tray was used forproducing the layered scaffolds. A CHA bone layer scaffold was producedand rehydrated as described in embodiment 1. 15.6 ml of the intermediatelayer slurry was pipetted on top of the rehydrated CHA bone layer. Thequantity added to each one can be varied to give an intermediated layerthickness of between 1 mm and 15 mm. The 2-layer construct was thenfreeze-dried as described in embodiment 1.

Cartilage Layer:

The cartilage layer slurry was produced by placing the Col1 slurry andCol2 slurry, produced as described above, into a beaker, placing thebeaker on ice and then blending until a homogenous solution wasproduced. The homogenous slurry was then degassed to remove air bubblesby placing the beaker in a vacuum chamber connected to a vacuum pump.The ratio of the Col1 slurry to the Col2 slurry (Col1:Col2) can varyfrom 0:1 to 1:0.

Prior to addition of the cartilage layer, the 2-layer scaffold wasrehydrated in acetic acid as previously described. The cartilage layerslurry was pipetted on top of the rehydrated 2-layer scaffold, thequantity used ranging from 3 ml to 60 ml, to give a scaffold rangingfrom 1 mm to 15 mm, depending on the thickness required. The entirestructure was freeze-dried again to produce a 3-layer scaffold.

Following freeze-drying the 3-layer porous structure was crosslinkedusing a dehydrothermal cross-linking process (DHT). This involvedplacing the structure in a vacuum oven (Fisher IsoTemp 201, FisherScientific, Boston, Mass.) to crosslink the collagen and thus provide anincrease in the mechanical strength of the structure. Cross-linking cancarried out at a temperature of from 105° C. to 180° C. under a vacuumof 50 mTorr for 24 hours.

Example 3

In another example, a 3-layered scaffold is produced where the baselayer is crosslinked via a chemical cross-linking method describedearlier (EDAC) prior to addition of the 2^(nd) layer in order to improvemechanical stiffness of the scaffold and maintain a equiaxed porestructure when additional layers are added to the scaffold. The degreeof cross-linking used can be controlled based on the structuralrequirements.

Example 4

Embodiment 4 relates to an alternative method for the production oflayered tissue engineering scaffolds. The process involves producing acollagen based slurry as above, and pipetting the 67.5 ml of the slurryinto a 127 mm×127 mm square 304 grade stainless steel tray. This tray isthen placed in the freeze-dryer and the slurry is frozen to atemperature of between −10° C. and −70° C. at a preferred constant rateof 0.9° C./min. This freezing rate can be varied between 0.1° C./min and10° C./min. The shelf temperature was then held constant for 60 minutesto complete the freezing process. The frozen scaffold was then removedfrom the freeze-dryer and 67.5 ml of a second slurry layer was appliedon top. The 2-layer structure was then freeze-dried. The cycle involvescooling the shelf to a temperature of between −10° C. and −70° C. at aconstant rate of 0.9° C./min. The shelf temperature was then heldconstant for 60 minutes to complete the freezing process. The shelftemperature was then ramped up to 0° C. over 160 minutes. The ice phasewas then sublimated under a vacuum of 200 mTorr at 0° C. for at least 17hours to produce the 2-layer porous scaffold.

Example 5

Embodiment 5 relates to a 3-layered scaffold and the method offabrication. The process involves the production of a collagen basedslurry as above, and pipetting 67.5 ml of the slurry into stainlesssteel tray as described above. The tray is placed into the freeze-dryerand the slurry is frozen to a temperature of between −10° C. and −70° C.at a suitable constant freezing rate, preferable 0.9° C./min. The shelftemperature was held constant for at least 60 minutes to complete thefreezing process. A second slurry layer was applied to this frozenslurry and the >60 minute freezing step was repeated. The frozen 2-layermaterial was then removed from the freeze-dryer and a 3^(rd)collagen-based slurry was again pipetted on top. This was then returnedto the freeze-drying and freeze-dried using a freeze-drying cycle wherethe shelf was cooled to a temperature of between −10° C. and −70° C. ata constant rate of 0.9° C./min. The shelf temperature was then heldconstant for 60 minutes to complete the freezing process. The shelftemperature was then ramped up to 0° C. over 160 minutes. The ice phasewas then sublimated under a vacuum of 200 mTorr at 0° C. for at least 17hours to produce a 3-layer scaffold.

Example 6

A further embodiment relating to the scaffold disclosed here relates tothe use of the scaffold as a growth factor delivery carrier system.Growth factors that could be incorporated into the scaffold include theTGF-β superfamily, IFG, FGF, BMP, PDGF, EGF and cannabinoids. Thesegrowth factors could be included into the scaffold in a number of ways,including by soaking the prepared scaffold in a solution containing thegrowth factor of interest, through cross-linking, using transcription,or through other methods.

Characterisation of Scaffolds

The properties of the individual scaffold layers and layered scaffoldsproduced in this study were compared to a control scaffold made of typeI collagen, fabricated using the standard protocol as detailed above.Briefly, a slurry was produced using 5 g/1 type I collagen in 0.05Macetic acid solution and lyophilised at a constant cooling rate to afinal freezing temperature of −40° C.

Mechanical Stiffness

In order to ensure survival following implantation the mechanicalproperties of the implant must be sufficient to withstand the forcesexperienced during load bearing. The mechanical properties of thescaffold have also been shown to affect cellular response (Engler etal.; 2006). The differentiation lineage for MSCs was found by Engler etal. to vary depending on substrate elasticity, with a neuronal phenotyperesulting on soft substrates and osteoblasts resulting on high stiffnesssubstrates. The mechanical properties would thus have particularimportance in applications where defect healing occurs due to theinfiltration of MSCs, for example, if the scaffold was to be used incombination with the microfracture technique. Mechanical testing wascarried out on 9.7 mm diameter samples using the Zwick Z050 MechanicalTesting Machine (Zwick/Roell, Germany). Prior to testing samples werepre-hydrated with phosphate buffered saline (PBS). The scaffolds wereloaded at a strain rate of 10% per minute and the modulus was defined asthe slope of a linear fit to the stress-strain curve over the 2-5%strain range.

The mechanical properties of each individual layer of the 3-layerscaffold produced in isolation and of the 3-layer scaffold weredetermined and compared to a standard collagen scaffold. The results areshown in FIG. 4. The bottom layer was found to have the highestcompressive modulus of approximately 0.95 kPa, significantly higher thanthe other two groups (p<0.05). This is due to the presence of the HAmineral phase. The compressive moduli of the intermediate layer and thetop layer were found to be between approximately 0.3 kPa and 0.4 kPa,with no significant difference being found between the two groups. Thecompressive modulus of the 3-layer scaffold was found to be similar tothat of the collagen scaffold, with no statistically significantdifference (p>0.05) being found between the two groups.

Distribution of Hydroxyapatite (HA)

The distribution of hydroxyapatite (HA) within a 2-layer scaffold wasinvestigated by embedding the scaffold in a polymer, carrying outhistological preparation and then staining with Toluidine blue stain.Microscopic analysis enabled HA distribution to be examined. Thepresence of HA particles within the collagen struts is evident, as shownin FIG. 1. X-ray diffraction (XRD) was used to analyse the effect of thefabrication process on the chemical composition of the bottom layer CHAscaffold. The XRD pattern for the bottom layer scaffold was compared tothat of the pure HA powder and to the standard XRD pattern for HA (JCPDS72-1243). The results, shown in FIG. 3, confirm the presence of HA inthe bottom layer CHA scaffold. No other calcium phosphate phases wereidentified, thus confirming that degradation of the HA component has notoccurred.

Scaffold Permeability

The permeability of a porous material is the ease with which a fluid canflow through it. High permeability is essential for tissue engineeredscaffold materials in order to allow cellular migration into theircentre. The permeability of the individual layers of the 3-layerscaffold is compared in FIG. 5. Scaffold permeability can be seen torelate to substrate stiffness, with the scaffolds which were found tohave a higher compressive modulus displaying the greatest permeability.The permeability of our 3-layer scaffold can be controlled by alteringthe mechanical properties of the individual scaffold layers, in order toproduce an optimal scaffold.

Porosity and Pore Structure

A high level of porosity is a vital requirement for scaffolds used fortissue regeneration in order to allow the infiltration of cells,diffusion of nutrients and removal of waste. If the porosity isinsufficient avascular necrosis will occur at the centre of theimplanted material, leading to failure of the construct. One of the mainadvantages of the present invention is the high degree of porositywithin all regions of the scaffold. The porosity of each of thecomponent layers of the 3-layer scaffold was determined using thedensity method as per F2450-04: Standard Guide for AssessingMicrostructure of Polymeric Scaffolds for Use in Tissue EngineeredMedical Products, using the following formulae

$V_{p} = {V_{T} - \frac{m_{s}}{\rho_{s}}}$%  Porosity = V_(p)/V_(T) × 100

Where Vp is the volume of the pores in the scaffold, V_(T) is the totalvolume of the scaffold, determined by measuring the sample dimensions,m_(s) is the mass of the scaffold, and ρ_(s) is the density of thematerial.

The average porosity of each layer is shown in FIG. 7. The top layer wasfound to have the highest porosity and the bottom layer the lowest. Thedifference between groups was found to be statistically significant(p<0.05). A reduction in porosity was seen due to the addition of HAparticles but this is negligible in real terms (99.5-98.8%). A highlevel of porosity is necessary in order to ensure the infiltration ofcells into the centre of the scaffold and also the supply of nutrientsand removal of waste from these cells. If porosity is insufficient,areas of necrosis will result within the scaffold.

The pore size and pore structure of scaffold materials is alsoimportant. A homogenous interconnecting pore structure with optimal poresize is necessary in order to successfully generate repair tissue. Ifpores are too small cell migration is limited, whereas if pores are toolarge there is a decrease in surface area, limiting cell adhesion(O'Brien F J, 2005; Murphy C M, 2009). One of the advantages of thefreeze-drying process used to produce the scaffolds detailed in thisinvention is the ability to precisely control pore size and porehomogeneity. The pore structure of the individual scaffold layers and of2-layer scaffolds was analysed by embedding the scaffold in JB4glycomethacrylate (Polysciences, Germany), in both longitudinal andtransverse plane, preparing the scaffolds histologically and stainingthem with toluidine blue prior to microscopic analysis. Representativemicrographs demonstrating the pore homogeneity of the individualscaffold layers are shown in FIG. 6. These micrographs demonstrate thatthe addition of type II collagen and hydroxyapatite to the variouslayers does not effect pore homogeneity.

Pore size was determined using a linear intercept method. The averagepore sizes of the individual scaffold layers produced in isolation usinga −40° C. freeze-drying cycle, are shown in FIG. 8. The average porediameters were found to range from 112 μm for the intermediate scaffoldto 136 μm for the bottom scaffold. The pore size of each individuallayer can be controlled by altering the freezing temperature used duringthe freeze-drying process.

A homogenous pore structure is also obtained when producing layeredscaffolds. The microscope images of sections from both the top andbottom layers of a 2-layer scaffold shown in FIG. 2 demonstrate thecapability of the ‘iterative layering’ method to produce a layeredconstruct with a highly porous structure and homogenous pore sizedistribution throughout.

Scanning Electron Microscopy (SEM) analysis of the 3-layered scaffold(FIG. 9) demonstrates the high degree of pore interconnectivitythroughout the construct. Structural continuity at the interfaces isevident, with the individual layers being seamlessly integrated. Thisseamless integration of the scaffold layers is vital in order to promotethe regeneration of anatomical repair tissue. This type of continuousstructure cannot be achieved using other layered scaffold productionmethods which involve for example the suturing or gluing together of thescaffold layers.

In-Vitro Bioactivity

The ability of cells to attach to, infiltrate through, and proliferatewithin the 3-layered scaffold described in this invention wasinvestigated through in vitro culture. Scaffold discs, 12.7 mm (½″) indiameter and 4 mm in height, were cut from pre-fabricated scaffoldsheets of the 3-layered scaffold material. The scaffolds were seededwith MC3T3-E1 mouse pre-osteoblast cells at a density of 2×10⁶ cells perscaffold. Scaffolds were evaluated at 7 and 14 days post seeding. FIG.12 shows transverse sections of a 3-layer scaffold, following 14 days inculture, prepared histiologically and stained using haematoxylin andeosin (H&E) staining. Cells were seen to infiltrate into scaffold andadhere to the collagen struts.

Cell number was determined by DNA quantification using Hoechst DNAassay. Qiazol Lysis Reagent was used to allow dissociation ofnucleoprotein complexes. Hoechst 33258 dye was then added tofluorescently label DNA and fluorescent emission was read using afluorescence spectrophotometer. Readings were converted to cell numberusing a standard curve. Cells numbers for the collagen and 3-layerscaffolds at 7 and 14 days are shown in FIG. 10. There was an increasein cell number of approximately 50% from day 7 to day 14 for both thecollagen and the 3-layer scaffolds, indicating that cells are readilyproliferating within both scaffold types. This demonstrates that thescaffolds are highly biocompatible, providing an excellent environmentfor the growth and differentiation of the MC3T3 cells.

Interfacial Adhesion Strength

The interfacial adhesion strength between the layers of the multi-layerconstruct described here is an important property. If adhesion strengthis insufficient delamination will occur at the layer interfaces.Interfacial adhesion strength of both 2-layer and 3-layer constructs wasdetermined using a custom designed rig fitted to the Zwick Z050Mechanical Testing Machine (Zwick/Roell, Germany). Testing involvedadhering the scaffold to test stubs using a high viscosity adhesive. Atensile load was applied to samples at a strain rate of 10% per minute.The samples were tested to failure. Pre-hydration of samples in PBS wascarried out prior to testing and testing was carried out in a bath ofPBS to maintain hydration during the test period. Fibre pullout wasobserved on the fracture surface following testing indicating trueintegration of the scaffold layers.

The invention is not limited to the embodiment hereinbefore describedwhich may be varied in construction and detail without departing fromthe spirit of the invention.

REFERENCES

-   1. Aigner T, Stove, 2003—Collagens—major component of the    physiological cartilage matrix, major target of cartilage    degeneration, major tool in cartilage repair, Advanced Drug delivery    Reviews 55 (2003) 1569-1593-   2. Newman A P, 1998—Newman A. P., (1998), Current Concepts,    Articular Cartilage Repair, The American Journal of Sports Medicine,    Vol. 26, No. 2-   3. Beris A E 2005—Beris A. E., et al. (2005), Advances in articular    cartilage repair, Injury, The International Journal of the Care of    the Injured, 36S, S14-S23-   4. Hunziker E B, 2001—Hunziker E B (2001) Growth-factor-induced    healing of partial-thickness defects in adult articular cartilage,    Osteoarthritis Cartilage 9: 22-32.-   5. Sherwood J K et al, 2002—Sherwood, J. K., (2002), A    three-dimensional osteochondral composite scaffold for articular    cartilage repair, Biomaterials 23 4739-4751-   6. Engler 2006—Engler A. J., Sen S., Sweeney H. L., Discher D. E.    Matrix Elasticity Directs Stem Cell Lineage Specification. Cell,    Volume 126, Issue 4, Pages 677-689. 2006-   Lee S H, Shin H: 2007—Lee, S. H., Shin, H., (2007), Matrices and    scaffolds for delivery of bioactive molecules in bone and cartilage    tissue engineering, Advanced drug delivery reviews, 59:339-359-   8. Miljkovic et al, 2008—Miljkovic N. et al. (2008) Chondrogenesis,    bone morphogenetic protein-4 and mesenchymal stem cells,    Osteoarthritis and Cartilage, Volume 16, Issue 10, Pages 1121-1130-   9. O'Brien F J, 2005—O'Brien F. J., Harley B. A., Yannas I. V., and    Gibson L. J. The effect of pore size on cell adhesion in    collagen-gag scaffolds. Biomaterials, 26:433-441, 2005-   10. Murphy C M, 2009—Murphy C. M., (2010) The effect of mean pore    size on cell attachment, proliferation and migration in    collagen-glycosaminoglycan scaffolds for bone tissue engineering,    Biomaterials, Volume 31, Issue 3, January 2010, Pages 461-466

What is claimed is:
 1. A cell seeded tissue engineering constructcomprising cells from a host seeded onto a multi-layer collagenscaffold, wherein the cells are disposed within pores of the multi-layerscaffold and the multi-layer scaffold comprises a three freeze-driedlayers in which an optional first layer comprises Type I collagen andHA; a second layer comprises Type I collagen, a Type II collagen, apolymer and/or biologic, and HA; and a third layer comprises Type Icollagen, Type II collagen, and a polymer and/or biologic.
 2. The cellseeded tissue engineering construct of claim 1, wherein the biologic isselected from the group consisting of a nucleic acid, a protein, apeptide, a cytokine, a hormone, a cell, and a growth factor.
 3. The cellseeded tissue engineering construct of claim 1, wherein the polymercomprises a glycosaminoglycan (GAG).
 4. A cell seeded tissue engineeringconstruct comprising cells from a host seeded onto a multi-layercollagen scaffold, wherein the cells are disposed within pores of themulti-layer scaffold and the multi-layer scaffold comprises a pluralityof freeze-dried layers in which an optional first layer consists ofcollagen and HA; a second layer consists essentially of a collagen, apolymer and/or biologic and HA; and a third layer consisting essentiallyof collagen, wherein a ratio of HA in the first layer to HA in thesecond layer is at least 1:1 (w/w), wherein the collagen component inthe first layer comprises Type I collagen, and the collagen component ofthe second and third layers comprises Type I and II collagen.
 5. Thecell seeded tissue engineering construct of claim 4, wherein the ratioof HA in the first layer to HA in the second layer is at least 3:1(w/w).
 6. The cell seeded tissue engineering construct of claim 4,wherein the scaffold has a continuous pore architecture extending acrossthe layers.
 7. The cell seeded tissue engineering construct of claim 6,wherein the porous scaffold layers are seamlessly integrated.
 8. Thecell seeded tissue engineering construct of claim 6, wherein scaffoldcomprises a pore architecture gradient extending across the layers.
 9. Acell seeded tissue engineering construct of claim 1, wherein themulti-layer scaffold comprises: (i) a plurality of porous freeze-driedlayers in which the scaffold has a continuous pore architectureextending across the layers, and in which the pore architecture of atleast two of the layers is typically different; (ii) a plurality ofporous freeze-dried layers in which the interface between each layer isseamless, and in which the pore architecture of at least two of thelayers is typically different; or (iii) continuous physical integrationat the interface of each adjacent layer, wherein each layer comprises apore architecture characteristic that is different to the other layers.10. A method for producing a cell seeded tissue engineering construct,the method comprising seeding cells from a host onto a multi-layercollagen scaffold and culturing the cells on the multi-layer scaffoldprior to implantation into a defect, wherein the cells are disposedwithin pores of the multi-layer scaffold and the multi-layer scaffoldcomprises: (i) a plurality of freeze-dried layers in which an optionalfirst layer comprises Type I collagen and HA; a second layer comprisesType I collagen, a Type II collagen, a polymer and/or biologic, and HA;and a third layer comprises Type I collagen, Type II collagen, and apolymer and/or biologic; (ii) a plurality of freeze-dried layers in anoptional first layer consists of collagen and HA; a second layer acollagen, a polymer and/or biologic and HA; and a third layer consistingessentially of collagen, wherein a ratio of HA in the first layer to HAin the second layer is at least 1:1 (w/w), wherein the collagencomponent in the first layer comprises Type I collagen, and the collagencomponent of the second and third layers comprises Type I and IIcollagen; (iii) a plurality of porous freeze-dried layers in which thescaffold has a continuous pore architecture extending across the layers,and in which the pore architecture of at least two of the layers istypically different; (iv) a plurality of porous freeze-dried layers inwhich the interface between each layer is seamless, and in which thepore architecture of at least two of the layers is typically different;or (v) continuous physical integration at the interface of each adjacentlayer, wherein each layer comprises a pore architecture characteristicthat is different to the other layers.
 11. The method of claim 10,further comprising preparing the multi-layer scaffold by an iterativelayering technique in which each layer is independently formed byfreezing, or lyophilisation followed by rehydration with an acidicsolvent, prior to addition of a following layer, wherein the final layeris formed by lyophilisation.
 12. The method of claim 11, wherein alllayers in the scaffold are formed by lyophilisation.
 13. The method ofclaim 10, further comprising cross-linking one or more of the layers ofthe scaffold.